Lidia Cerquetti

Rosiglitazone induces autophagy in H295R and cell cycle deregulation in SW13 adrenocortical cancer cells

Thiazolidinediones, specific peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands, used in type-2 diabetes therapy, show favourable effects in several cancer cells. In this study we demonstrate that the growth of H295R and SW13 adrenocortical cancer cells is inhibited by rosiglitazone, a thiazolidinediones member, even though the mechanisms underlying this effect appeared to be cell-specific. Treatment with GW9662, a selective PPAR-γ-inhibitor, showed that rosiglitazone acts through both PPAR-γ-dependent and -independent mechanisms in H295R, while in SW13 cells the effect seems to be independent of PPAR-γ. H295R cells treated with rosiglitazone undergo an autophagic process, leading to morphological changes detectable by electron microscopy and an increased expression of specific proteins such as AMPKα and beclin-1. The autophagy seems to be independent of PPAR-γ activation and could be related to an increase in oxidative stress mediated by reactive oxygen species production with the disruption of the mitochondrial membrane potential, triggered by rosiglitazone. In SW13 cells, flow cytometry analysis showed an arrest in the G0/G1 phase of the cell cycle with a decrease of cyclin E and cdk2 activity, following the administration of rosiglitazone. Our data show the potential role of rosiglitazone in the therapeutic approach to adrenocortical carcinoma and indicate the molecular mechanisms at the base of its antiproliferative effects, which appear to be manifold and cell-specific in adrenocortical cancer lines.

Increased metastatic lymph node 64 and CYP17 expression are associated with high stage prostate cancer.

The metastatic lymph node 64 (MLN64), which is localized in the human chromosome 17, encodes a protein with strong homology with steroidogenic acute regulatory protein. Its overexpression in human breast carcinomas and MLNs led to the hypothesis that this protein could be involved in intraneoplastic steroidogenesis. In the present study, we investigated the expression of MLN64 in prostate cancer, another hormone-dependent tumor, and compared its expression with that of CYP17, the gene encoding for the key enzyme of androgen synthesis. We investigated by RT-PCR the expression of MLN64 and CYP17 in 60 prostatic tumors and compared their expression with the stage of disease and the appearance of relapses in a follow-up of 24 months. We found MLN64 and CYP17 expressed in all samples examined, with significantly higher expression in neoplastic tissues with respect to normal tissues (NTs). Moreover, only in neoplastic but not in NTs, a positive linear correlation was found between MLN64 and CYP17 gene expression. MLN64 and CYP17 expression seems to correlate with high stage, high Gleason score and short relapse-free time. These data, for the first time, demonstrate the presence of MLN64 and CYP17 expression in both normal and neoplastic prostatic tissues. The biological role of MLN64 in human prostate and, particularly, in neoplastic tissue is still unclear. Our findings concerning MLN64 and CYP17 gene expression and their significant positive correlation in human prostate cancer may suggest their possible role in intraneoplastic autonomous steroidogenesis.

Targeting Estrogen Receptor-α Reduces Adrenocortical Cancer (ACC) Cell Growth in Vitro and in Vivo: Potential Therapeutic Role of Selective Estrogen Receptor Modulators (SERMs) for ACC Treatment

CONTEXT Adrenocortical carcinoma (ACC) is a rare tumor with a very poor prognosis and no effective treatment. ACC is characterized by an increased production of IGF-II and by estrogen receptor (ER)-α up-regulation. OBJECTIVE The objective of this study was to define the role played by ERα in 17β-estradiol (E2)- and IGF-II-dependent ACC growth and evaluate whether selective estrogen receptor modulators are effective in controlling ACC growth in vivo. EXPERIMENTAL DESIGN The human adrenocortical cell line H295R was used as an in vitro model and to generate xenograft tumors in athymic nude mice. RESULTS In H295R cells IGF-II controlled expression of steroidogenic factor-1 that, in turn, increased aromatase transcription and, consequently, estrogen production, inducing cell proliferation. ERα silencing significantly blocked E2- and IGF-II-dependent cell proliferation. This effect was dependent on the regulation of cyclin D1 expression by ERα, activated in response to both E2 and IGF-II. In fact, IGF-II induced ERα activation by phosphorylating serine 118 and 167. Furthermore, we demonstrated that ERα mediated E2-induced nongenomic signaling that stimulated IGF-I receptor (IGF1R), ERK1/2, and AKT phosphorylation, resulting in a ligand-independent activation of the IGF1R-induced pathway. In addition, E2 potentiated this pathway by up-regulating IGF1R expression as a consequence of increased cAMP-responsive element binding protein activation and binding to IGF1R promoter. The estrogen antagonist, hydroxytamoxifen, the active metabolite of tamoxifen, reduced IGF1R protein levels and both E2- and IGF-II-induced cell proliferation. Moreover, H295R xenograft growth was strongly reduced by tamoxifen. CONCLUSION These findings establish a critical role for ERα in E2- and IGF-II-dependent ACC proliferation and provide a rationale for targeting ERα to control the proliferation of ACC.

Proteomic profiles in hyperandrogenic syndromes.

Background: Polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH) represent the most common causes of hyperandrogenism. Although the etiopathogeneses of these syndromes are different, they share many clinical and biochemical signs, such as hirsutism, acne, and chronic anovulation. Experimental data have shown that peripheral T-lymphocytes function as molecular sensors, being able to record molecular signals either at staminal and mature cell levels, or hormones at systemic levels. Methods: Twenty PCOS women and 10 CAH with 21-hydroxylase deficiency, aged between 18–35 yr, were studied. T-cells purified from all patients and 20 healthy donors have been analyzed by 2-dimensional gel electrophoresis. Silver-stained proteomic map of each patient was compared with a control map obtained by pooling protein samples of the 20 healthy subjects. Results: Spots of interest were identified by peptide mass fingerprint. Computer analysis evidenced several peptidic spots significantly modulated in all patients examined. Some proteins were modulated in both syndromes, others only in PCOS or in CAH. These proteins are involved in many physiological processes as the functional state of immune system, the regulation of the cytoskeleton structure, the oxidative stress, the coagulation process, and the insulin resistance. Conclusion: Identification of the physiological function of these proteins could help to understand ethiopathogenetic mechanisms of hyperandrogenic syndromes and its complications.

p53 Stabilization Induces Cell Growth Inhibition and Affects IGF2 Pathway in Response to Radiotherapy in Adrenocortical Cancer Cells

Adrenocortical carcinoma (ACC) is a very rare endocrine tumour, with variable prognosis, depending on tumour stage and time of diagnosis. However, it is generally fatal, with an overall survival of 5 years from detection. Radiotherapy usefulness for ACC treatment has been widely debated and seems to be dependent on molecular alterations, which in turn lead to increased radio-resistance. Many studies have shown that p53 loss is an important risk factor for malignant adrenocortical tumour onset and it has been reported that somatic mutations in TP53 gene occur in 27 to 70% of adult sporadic ACCs. In this study, we investigated the role of somatic mutations of the TP53 gene in response to ionizing radiation (IR). We studied the status of p53 in two adrenocortical cell lines, H295R and SW-13, harbouring non-functioning forms of this protein, owing to the lack of exons 8 and 9 and a point mutation in exon 6, respectively. Moreover, these cell lines show high levels of p-Akt and IGF2, especially H295R. We noticed that restoration of p53 activity led to inhibition of growth after transient transfection of cells with wild type p53. Evaluation of their response to IR in terms of cell proliferation and viability was determined by means of cell count and TUNEL assay.wtp53 over-expression also increased cell death by apoptosis following radiation in both cell lines. Moreover, RT-PCR and Western blotting analysis of some p53 target genes, such as BCL2, IGF2 and Akt demonstrated that p53 activation following IR led to a decrease in IGF2 expression. This was associated with a reduction in the active form of Akt. Taken together, these results highlight the role of p53 in response to radiation of ACC cell lines, suggesting its importance as a predictive factor for radiotherapy in malignant adrenocortical tumours cases.

Human placenta-derived neurospheres are susceptible to transformation after extensive in vitro expansion

IntroductionThe cancer stem cell model links neoplastic cells with normal stem cell biology, but little is known on how normal stem cells are transformed into cancer stem cells.MethodsTo investigate the processes underlying the transformation of normal stem cells we developed in vitro a cancer stem cell model from human amniotic and chorionic placenta membranes. In this model we studied the expression of specific stem cell molecules by flow cytometry, and genes, by real time RT-PCR. Microscopy immunfluorescence was employed to investigate the proliferative and differentiation patterns. Fluorescence microscopy and FACS were employed to investigate the proliferative and differentiation patterns. To evaluate the tumorigenic potential of our model we injected the cells into NOD.CB17-Prkdcscid/NCrHsd mice.ResultsNormal human stem cells from amniotic and chorionic placenta membranes were converted into neural cell lineages, under specific conditions, to form secondary neurospheres with a capacity for self-renewal. After extensive in vitro culture, these cells underwent spontaneous transformations and acquired a neuroblastoma (NB)-like phenotype with an elevated proliferative potential that is comparable to established neuroblastoma cell lines. The ability of these cells to transform their phenotype was evidenced by increased clonogenic ability in vitro; by augmented expression level of certain proliferation- and transformation-related genes (e.g., CCNA2, MYCN, ENPP2, GRIA3, and KIT); by the presence of multinucleated and hyperdiploid cells. We further demonstrated that the transformed phenotype is an NB by measuring the expression of NB-specific markers, disialoganglioside GD2 and N-Myc proteins.ConclusionsWe have developed a cancer stem cell model starting from normal human stem cells derived from amniotic and chorionic placenta membranes. These cells are able to differentiate into neural cell lineages and to undergo spontaneous transformations and acquire an NB-like phenotype.

Effect of mitotane on mouse ovarian follicle development and fertility

Mitotane (MTT) is an adrenolytic drug used in advanced and adjuvant treatment of adrenocortical carcinoma, in Cushings disease and in ectopic syndrome. However, knowledge about its effects on the ovary is still scarce. The purpose of this study is to investigate the effect of MTT on the ovary using in vivo and in vitro models. The study was performed in CD1 mice and in the COV-434 human ovarian granulosa cell line. We examined ovarian morphology, follicle development, steroidogenesis and procreative function in mice and the effect of MTT on cell growth in vitro Our results revealed that treatment of CD1 mice with MTT induces a decrease in early antral follicles with a subsequent increase in the secondary follicles, measured by the increased levels of anti-Mullerian Hormone (P < 0.05) and decreased levels of FSH receptor (P < 0.05). Moreover, we observed a significant decrease in Cyp11a1 (P < 0.01) and Cyp17a1 (P < 0.001) mRNA level in MTT-treated animals. Ovulation, induced by PMSG/hCG stimulation, was also significantly impaired, with a reduction in the number of ovulated oocytes (P < 0.01) and fewer corpora lutea in treated animals. Likewise, the mating experiment demonstrated a delay in the time of conception as well as fewer pups per litter in MTT-treated mice (P < 0.05). Experiments performed on the COV-434 cell line showed a significant inhibition of growth followed by apoptosis (P < 0.01). In conclusion, our study highlights the key points of ovarian folliculogenesis affected by MTT and demonstrates impairment of the ovulation process with a negative impact on conception, which is nevertheless preserved.

C-MYC modulation induces responsiveness to paclitaxel in adrenocortical cancer cell lines.

C-MYC is overexpressed in many types of cancer linked to poor prognosis. We examined the c-Myc protein expression in adrenocortical cancer (ACC) cells to investigate the role of this protein in the neoplasm, its involvement in chemotherapy and finally to determine whether c-Myc could be considered a prognostic factor in patients with ACC. H295R and SW13 cell lines were treated with paclitaxel. c-Myc overexpressing cell clones were achieved by transfecting the H295R cell line with the pcDNA3-hMYC plasmid expressing the full-lengh C-MYC coding sequence. The SW13 cell line was transfected with siRNA oligonucleotides for C-MYC. Cell cycle analysis was evaluated by flow cytometry. c-Myc, cyclin B1 and pro caspase expression levels were evaluated by western blot analysis. We found that expression of c-Myc was highly expressed in the SW13 cells, whereas the protein was undetectable in the H295R cells. Different doses of paclitaxel were required in the two ACC cell line to induce a block in the G2 phase, characterized by increased cyclin B1 levels and to induce apoptosis by pro-caspase-3 activation. Interestingly, the silencing of C-MYC mRNA prevented paclitaxel induced apoptosis in SW13 cells, whereas in the H295R cells the overexpression of C-MYC rendered the cells more prone to growth inhibition after paclitaxel exposure. The present study directly demonstrates that C-MYC plays a central role in controlling proliferation in ACC cells after paclitaxel treatment and that c-Myc could be considered as a marker for predicting response to chemotherapeutic agents in ACC cell lines.

Il trattamento farmacologico della sindrome di Cushing a etiologia surrenalica

ACTH-independent adrenal Cushings syndrome is the least common form of endogenous hypercortisolism. Recently, advances in genetics have allowed the description of several forms different to pathogenetic etiology, morphostructural characteristics and evolution towards the hypercortisolism. Alongside these, the adrenocortical carcinoma is also frequently responsible of a hypercortisolism clinical picture. The availability of steroidogenesis inhibitors, such as metyrapone and ketoconazole, provides to endocrinologist a therapeutic chance against different metabolic disorders sustained by hypercortisolism. Mitotane, an adrenolitic compound, is used alone in adjuvant therapy or in combination with different chemotherapy drugs in the treatment of adrenocortical carcinoma and in the treatment of severe Cushings syndrome.